Anaerobe 54 (2018) 8e18

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Anaerobe

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Research paper Anaerobes in the microbiome The impact of butyricum MIYAIRI 588 on the murine gut microbiome and colonic tissue

Mao Hagihara a, Rieko Yamashita a, Asami Matsumoto a, b, Takeshi Mori b, Yasutoshi Kuroki b, c, Hayami Kudo c, Kentaro Oka b, c, Motomichi Takahashi b, c, * Tsunemasa Nonogaki d, Yuka Yamagishi b, Hiroshige Mikamo b, a Department of Molecular Epidemiology and Biomedical Sciences, Aichi Medical University, Japan b Department of Clinical Infectious Diseases, Aichi Medical University, Japan c Miyarisan Pharmaceutical Co., Ltd, Japan d Department of Pharmacy, College of Pharmacy, Kinjyo Gakuin University, Japan article info abstract

Article history: Background: Clostridium butyricum MIYAIRI 588 (CBM 588) is a bacterium that is used as an Received 28 February 2018 anti-diarrheal medicine in Japan. However, the impact of this probiotic on the gut microbiome has not Received in revised form been fully elucidated, especially, when used with antimicrobials. 26 July 2018 Material and methods: In an in vivo study, CBM 588 monotherapy, clindamycin monotherapy, CBM 588 Accepted 30 July 2018 and clindamycin (combination therapy), or normal saline (control) was orally administered to mice for 4 Available online 1 August 2018 days, and fecal samples were collected for 18 days to enumerate C. butyricum. We also extracted DNA Handling editor: Kaori Tanaka from these fecal samples for metagenomics analysis by amplification of the V3-V4 region of the bacterial 16S rRNA gene and MiSeq Illumina sequencing. In addition, the concentrations of some short chain fatty Keywords: acids were assessed in the fecal samples. A histological analysis was also conducted. Clostridium butyricum Results: On day 4 (the last treatment day), there was no difference in the total counts of C. butyricum Clindamycin between the CBM 588 monotherapy and combination therapy groups (5.21 ± 0.78 vs. 5.13 ± 0.45 Microbiome log10 cfu/g, p ¼ 0.86). Clindamycin treatment resulted in dramatic increases in the phylum , especially Enterobacteriaceae, , Lactobacillus, and Enterococcus, compared with the other groups during the treatment period. CBM 588 treatment modified the bacterial community composition at lower phylogenetic levels. Some bacterial taxa, such as Bifidobacterium, Coprococcus, and Bacteroides, were significantly increased in the combination therapy group when compared with the other groups. In the metabolic analysis, CBM 588 enhanced lactic acid production. It also enhanced the efficiency of lactic acid use for the production of . Only the clindamycin monotherapy group showed abnormal colon tissue, with superficial epithelial necrosis and the presence of inflammatory cells. Conclusion: CBM 588 treatment modulated the gut microbiota composition under dysbiosis due to the use of an antimicrobial with strong activity against anaerobes and significantly reduced epithelial damage. © 2018 Elsevier Ltd. All rights reserved.

1. Introduction between 5% and 25% among antimicrobials [1]. The cause of AAD is disturbance of the composition of the normal intestinal flora, Diarrhea is a frequent adverse effect of antimicrobial use. The leading to the overgrowth of pathogenic , and the incidence of antimicrobial-associated diarrhea (AAD) varies allergic and toxic effects of the antimicrobial on the intestinal mucosa or pharmacologic effects on intestinal motility. Thus, pro- biotics have been used for the treatment and prophylaxis of AAD [2,3]. * Corresponding author. Department of Clinical, Infectious Diseases, Aichi Med- ical University Hospital, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan. Probiotics affect the interaction between resident microorgan- E-mail address: [email protected] (H. Mikamo). isms and the physiological functions of the host. Clostridium https://doi.org/10.1016/j.anaerobe.2018.07.012 1075-9964/© 2018 Elsevier Ltd. All rights reserved. M. Hagihara et al. / Anaerobe 54 (2018) 8e18 9 butyricum MIYAIRI 588 (CBM 588) is probiotic bacterium that is 500 mg/kg per day (3.4 108 cfu/kg per day). Clindamycin was also used as an anti-diarrheal medicine in Japan. CBM 588 has been administered by oral gavage at 40 mg/kg per day. CBM 588 powder reported to both prevent and cure diarrhea [4e6]. C. butyricum is a was dissolved in sterilized water to prepare a suspension. After strictly anaerobic Gram-positive and a common human mixing the suspension sufficiently, the mice were forced to drink it and animal gut commensal bacterium that is also frequently found by using sonde. For the combination group, CBM 588 and clinda- in the environment. C. butyricum can produce , which is mycin were dissolved in sterilized water separately. A suspension a key characteristic related to its high tolerance to antimicrobials containing half the daily dose was administered twice a day, at 10 and gastric acid in the intestinal tract. Thus, CBM 588 may be not a.m. and 4 p.m., for 4 days. affected by antimicrobials, which may provide an advantage over Assessment of physiological condition. Weight loss and stool other probiotics. However, few studies have evaluated the effects of consistency were assessed daily to determine any physical changes. CBM 588 on the human microbiome, especially with concomitant Body weights were recorded every other day. These data are re- administration of an antimicrobial that has activity against ported as the percentage of weight lost compared to the initial body anaerobes. weight. The intestinal tract harbors a complex microbial community Enumerating C. butyricum in feces. C. butyricum was that plays a key role in nutrition and health. The intestinal micro- enumerated in samples obtained before treatment (day 0), on the biome has a symbiotic relationship with the host. In certain age second (day 2) and fourth (day 4) days of treatment, and on the categories, the microbial diversity in the intestine remains stable in second (day 6), fourth (day 8), sixth (day 10), eighth (day 12), 10th healthy individuals [7]. However, a shift in the microbial compo- (day 14), 12th (day 16), and 14th (day 18) days after treatment sition, referred to as dysbiosis, has been described in several pa- termination. On each sampling day, at least 0.3 g of feces were thologies, and alternations of the gut microbiome are believed to collected and placed into 0.45 ml of transport medium. underlie the development of AAD. Such changes in the gut micro- To determine the concentration of fecal C. butyricum, biome allow the outgrowth of certain pathogens, such as C. difficile C. butyricum selective medium was used [11]. The fecal specimens and C. perfringens [8,9]. were serially diluted (10 fold) to 105 with culture medium, spread The gut microbiome also aids in digestion. Alteration of the gut on the selective agar plates, and incubated for 24 h. Then, the microbiome can lead to significant changes in the colonic envi- C. butyricum colonies were counted. The viable count, that is the ronment caused by variation in the concentration and distribution number of colonies growing on the plates, was converted to the of organic compounds, such as carbohydrates, short chain fatty number of per gram of feces. Additionally, we exposed acids (SCFAs), and bile acids [3]. In a previous in vitro study simu- undiluted fecal specimens (0.05 ml) to ethanol. Then, the same lating the luminal contents of the proximal colon, clindamycin process was used to determine the numbers of C. butyricum spores reduced the number of anaerobes (Clostridium and Bacteroides in the fecal samples. The detection limit was 2.0 (log10 cfu) per gram species). This led to a reduction in the concentration of SCFAs [10]. of feces. Various metabolites produced by the resident microbiome play a DNA extraction. To characterize the microbiome composition in significant role in host physiology. Hence, decreased bacterial car- the colon, fecal samples from each mouse were analyzed by bohydrate metabolism in the colonic mucosa may result in a sequencing the V3eV4 regions of the 16S rRNA gene. DNA extrac- functional disturbance. tion and 16S rRNA sequencing were conducted as previously Despite the importance of the gut microbiome, there are few described [12]. The fecal contents from the mice were immediately reports on the modulating effects of CBM 588 on the gut micro- suspended in buffer containing 4 M guanidium thiocyanate, biome and metabolism. In this study, we examined the effects of 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA (pH 8.0) and stored at CBM 588 on the murine intestinal microbiome through multi- 4 C until use. For use, the fecal pellets were suspended in 10 mM omics approaches (genomics and metabolomics), and evaluated Tris-HCl and 10 mM EDTA buffer (pH 8.0). Next, lysozyme (final the histopathological changes to explain the mechanisms under- concentration, 15 mg/mL; Sigma) was added, and the samples were lying the effects of CBM 588. incubated at 37 C for 1 h. Then, purified achromopeptidase (Wako) was added to a final concentration of 2000 U/mL and incubated at 2. Material and methods 37 C for 30 min. Next, SDS (final concentration: 1%) was added to the cell suspension and mixed well. Then, proteinase K (Merck) was Material. CBM 588 bacterial powder was used for the in vivo added (final concentration: 1 mg/mL) to the suspension, and the studies. It is composed of 2.2 1010 cfu/g (Lot 61GT; MIYARISAN mixture was incubated at 55 C for 1 h. High-molecular mass DNA Pharmaceutical Co., Ltd.). Clindamycin for injection was purchased was isolated and purified by phenol/chloroform extraction, ethanol from Pfizer Japan, Inc.. Immediately before each in vivo experiment, precipitation, and finally polyethylene glycol precipitation. the CBM 588 powder was weighed and reconstituted with sterile Gut microbiome analysis. Meta 16S rRNA Gene Sequencing PCR water. Clindamycin was diluted with appropriate diluents to ach- was performed by using Ex Taq Hot Start (TAKARA) and the Illu- ieve the desired concentration. Clindamycin solution was stored mina forward primer 50-AATGATACGGCGACCACCGAGATCTACAC under refrigeration and discarded 12 h after reconstitution. (adaptor sequence) þ barcode (eight bases) þ ACACTCTTTCCC Animals and housing. Specific-pathogen-free, female ICR mice TACACGACGCTCTTCCGATCT (sequence primer) þ CCTACGGGNG (9e10 weeks old) weighing approximately 30 g were obtained from GCWGCAG-30 (341F) and the Illumina reverse primer 50-CAAG- Charles River Laboratories Japan, Inc. The mice were maintained CAGAAGACGGCATACGAGAT (adaptor sequence) þ barcode (eight and utilized according to National Research Council recommen- bases) þ GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (sequence dations and were provided food and water ad libitum. The study primer) þ GACTACHVGGGTATCTAATCC-30 (805R) to amplify the was reviewed and approved by the ethics committee of Aichi hypervariable V3eV4 region of the 16S rRNA gene. Amplicons Medical University. generated from each sample were subsequently purified using SPRI Experimental set up. Twenty female ICR mice were divided into select (Beckman Coulter). The amount of DNA was quantified using four treatment groups as follows (n ¼ 5): (i) control group, (ii) the QuantiFluor dsDNA System and a Quantus Fluorometer clindamycin monotherapy group, (iii) CBM 588 monotherapy (Promega). Mixed samples were prepared by pooling approxi- group, and (iv) combination therapy group (CBM mately equal amounts of each amplified DNA and sequenced using 588 þ clindamycin). CBM 588 was administered by oral gavage at the MiSeq Reagent Kit V3 (600 cycle) and a MiSeq sequencer Download English Version: https://daneshyari.com/en/article/8744491

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